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Quantitative analysis of single-molecule force spectroscopy on folded chromatin fibers.

Meng H, Andresen K, van Noort J - Nucleic Acids Res. (2015)

Bottom Line: Comparison of force-extension curves between single nucleosomes and chromatin fibers shows that embedding nucleosomes in a fiber stabilizes the nucleosome by 10 kBT.Chromatin fibers with 20- and 50-bp linker DNA follow a different unfolding pathway.These results have implications for accessibility of DNA in fully folded and partially unwrapped chromatin fibers and are vital for understanding force unfolding experiments on nucleosome arrays.

View Article: PubMed Central - PubMed

Affiliation: Biological and Soft Matter Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.

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Related in: MedlinePlus

Detailed analysis of the unfolding of a single chromatin fiber. (A) A zoom in on the high-force region shows discrete steps in extension. Dashed gray lines represent the extensions of all states that are composed of extended and fully unwrapped nucleosomes. The fit match was obtained for ext = 4.6 nm. The black line shows the best match between individual data points and the various states of unwrapping. (B) Step size distribution of the data shown in (A) obtained from a 10-point window t-test analysis. (C) Unfolding of a 15*197 NRL chromatin fiber at low force. Below 7 pN the extension starts to deviate from a string of extended nucleosomes (gray dashed lines). A single transition (black dashed line) does not capture the force-extension data. The black line shows a fit to Equation (8), while constraining Lwrap = 89 bp and ext = 4.6 nm, yielding = 21.2±0.1 kBT, ΔG2= 4.3±0.1 kBT. (D) The corresponding probability for a nucleosome to be in a fiber (low force), a single wrap (intermediate force) or in the extended conformation (high force).
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Figure 3: Detailed analysis of the unfolding of a single chromatin fiber. (A) A zoom in on the high-force region shows discrete steps in extension. Dashed gray lines represent the extensions of all states that are composed of extended and fully unwrapped nucleosomes. The fit match was obtained for ext = 4.6 nm. The black line shows the best match between individual data points and the various states of unwrapping. (B) Step size distribution of the data shown in (A) obtained from a 10-point window t-test analysis. (C) Unfolding of a 15*197 NRL chromatin fiber at low force. Below 7 pN the extension starts to deviate from a string of extended nucleosomes (gray dashed lines). A single transition (black dashed line) does not capture the force-extension data. The black line shows a fit to Equation (8), while constraining Lwrap = 89 bp and ext = 4.6 nm, yielding = 21.2±0.1 kBT, ΔG2= 4.3±0.1 kBT. (D) The corresponding probability for a nucleosome to be in a fiber (low force), a single wrap (intermediate force) or in the extended conformation (high force).

Mentions: The discrete steps in extension at forces above 6 pN represent the sequential unwrapping of the last DNA from each nucleosome and have been studied abundantly with optical tweezers (4,27) and MTs (51). MTs act as a force clamp rather than a position clamp, resulting in a staircase-like force-extension curve instead of the typical saw-tooth pattern obtained with optical tweezers. Figure 3A shows a zoom in on these high-force transitions. The corresponding step size distribution is shown in Figure 3B. A step size of 22 ± 3 nm was found, in range with previous studies on various DNA substrates and under different buffer conditions.


Quantitative analysis of single-molecule force spectroscopy on folded chromatin fibers.

Meng H, Andresen K, van Noort J - Nucleic Acids Res. (2015)

Detailed analysis of the unfolding of a single chromatin fiber. (A) A zoom in on the high-force region shows discrete steps in extension. Dashed gray lines represent the extensions of all states that are composed of extended and fully unwrapped nucleosomes. The fit match was obtained for ext = 4.6 nm. The black line shows the best match between individual data points and the various states of unwrapping. (B) Step size distribution of the data shown in (A) obtained from a 10-point window t-test analysis. (C) Unfolding of a 15*197 NRL chromatin fiber at low force. Below 7 pN the extension starts to deviate from a string of extended nucleosomes (gray dashed lines). A single transition (black dashed line) does not capture the force-extension data. The black line shows a fit to Equation (8), while constraining Lwrap = 89 bp and ext = 4.6 nm, yielding = 21.2±0.1 kBT, ΔG2= 4.3±0.1 kBT. (D) The corresponding probability for a nucleosome to be in a fiber (low force), a single wrap (intermediate force) or in the extended conformation (high force).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Detailed analysis of the unfolding of a single chromatin fiber. (A) A zoom in on the high-force region shows discrete steps in extension. Dashed gray lines represent the extensions of all states that are composed of extended and fully unwrapped nucleosomes. The fit match was obtained for ext = 4.6 nm. The black line shows the best match between individual data points and the various states of unwrapping. (B) Step size distribution of the data shown in (A) obtained from a 10-point window t-test analysis. (C) Unfolding of a 15*197 NRL chromatin fiber at low force. Below 7 pN the extension starts to deviate from a string of extended nucleosomes (gray dashed lines). A single transition (black dashed line) does not capture the force-extension data. The black line shows a fit to Equation (8), while constraining Lwrap = 89 bp and ext = 4.6 nm, yielding = 21.2±0.1 kBT, ΔG2= 4.3±0.1 kBT. (D) The corresponding probability for a nucleosome to be in a fiber (low force), a single wrap (intermediate force) or in the extended conformation (high force).
Mentions: The discrete steps in extension at forces above 6 pN represent the sequential unwrapping of the last DNA from each nucleosome and have been studied abundantly with optical tweezers (4,27) and MTs (51). MTs act as a force clamp rather than a position clamp, resulting in a staircase-like force-extension curve instead of the typical saw-tooth pattern obtained with optical tweezers. Figure 3A shows a zoom in on these high-force transitions. The corresponding step size distribution is shown in Figure 3B. A step size of 22 ± 3 nm was found, in range with previous studies on various DNA substrates and under different buffer conditions.

Bottom Line: Comparison of force-extension curves between single nucleosomes and chromatin fibers shows that embedding nucleosomes in a fiber stabilizes the nucleosome by 10 kBT.Chromatin fibers with 20- and 50-bp linker DNA follow a different unfolding pathway.These results have implications for accessibility of DNA in fully folded and partially unwrapped chromatin fibers and are vital for understanding force unfolding experiments on nucleosome arrays.

View Article: PubMed Central - PubMed

Affiliation: Biological and Soft Matter Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.

Show MeSH
Related in: MedlinePlus